Parahydrogen-induced polarization allows 2000-fold signal enhancement in biologically active derivatives of the peptide-based drug octreotide

Octreotide, a somatostatin analogue, has shown its efficacy for the diagnostics and treatment of various types of cancer, i.e., in octreotide scan, as radio-marker after labelling with a radiopharmaceutical. To avoid toxicity of radio-labeling, octreotide-based assays can be implemented into magnetic resonance techniques, such as MRI and NMR. Here we used a Parahydrogen-Induced Polarization (PHIP) approach as a cheap, fast and straightforward method. Introduction of l-propargyl tyrosine as a PHIP marker at different positions of octreotide by manual Solid-Phase Peptide Synthesis (SPPS) led to up to 2000-fold proton signal enhancement (SE). Cell binding studies confirmed that all octreotide variants retained strong binding affinity to the surface of human-derived cancer cells expressing somatostatin receptor 2. The hydrogenation reactions were successfully performed in methanol and under physiologically compatible mixtures of water with methanol or ethanol. The presented results open up new application areas of biochemical and pharmacological studies with octreotide.


Reversed Phase High-Performance Liquid Chromatography (RP-HPLC)
Reversed phase high-performance liquid chromatography (RP-HPLC) for analytical purposes was conducted using a Waters HPLC setup consisting of a Waters Alliance e2695 equipped with a Waters 2998 PDA detector. The detection wavelength was chosen depending on the analyte between 214, 254, 280 and 301 nm. The eluent system for the HPLC system comprised eluent A (0.1% aq. TFA) and eluent B (99.9% acetonitrile and 0.1% TFA). Unless otherwise specified, analytical HPLC runs were conducted at a flow rate of 1 ml/min with a t gradient of 20% to 80% of eluent B over 20 min. For the analysis, a Nucleosil 100-5 C18 column from Macherey-Nagel (5 μm, 100 Å) was used. Preparative isolation of the peptide was performed on a Knauer Multokrom RP18 column 20×250 mm (5 μm, 100 Å) employing a flow rate of 9 ml/min and an isocratic elution, namely,40 % ACN in 0.1 % aqueous TFA over the course of 60 min.

Electrospray Mass Spectrometry (ESI-MS)
Electron Spray Ionization (ESI) mass spectra were recorded with a Bruker Impact II mass spectrometer.

Flash Chromatography
Flash chromatography was conducted on a Büchi Pure C815 Flash using hexane (A) and ethyl acetate (B) as solvents and a Büchi FlashPure Select 12 g Silica 15 μm column. The raw product was loaded by solid loading on silica. For this the substrate was dissolved in DCM, mixed with silica gel 60 and the solvent evaporated under reduced pressure.

Synthesis of Fmoc-Thr(tBu)-ol
Fmoc-Thr(tBu)-ol was obtained by reduction of threonine Fmoc-Thr(tBu)-OH using NaBH4 in an organic/aqueous medium according to the procedure described by Rodriguez et al. 1 To a solution of Fmoc and tertbutyl-protected threonine (1.5 g, 3.77 mmol) in THF, (15 ml), cooled on an ice-salt bath, Nmethyl morpholine (420 µl, 3.78 mmol) and isobutyl chloroformate (490 µl, 3.77 mmol) were successively added. After one minute a solution of sodium borohydride (580 mg, 15.33 mmol) in water (2.1 ml) was added at once, producing a strong evolution of gas, followed by water (200 ml) 30 seconds afterwards. The cloudy suspension was extracted with DCM two times. Centrifugation was used to aid the phase separation. The organic phase was collected and evaporated in a rotary evaporator (40 °C, 450 mbar), yielding a clear viscous raw product (1.47 g for the first, and 1.67 g for the second batch). The first batch was used without further treatment. Cleanup of the second batch's raw product was performed by flash chromatography (

Synthesis of Fmoc-TTDS Spacer
The synthesis of Fmoc-TTDS was carried out following the procedure described by Z. G. Zhao et al. 2 A solution of 11.02 g (50 mmol) 4,7,10-trioxa-1,13-tridecanediamine in 200 ml acetonitrile was cooled to 0 °C while adding 5 g (50 mmol) of succinic anhydride in 200 ml acetonitrile dropwise in the period of one hour. The formed precipitate was separated from the supernatant solution and redissolved in 500 ml of 50 % (v/v) acetonitrile in water. Again, under cooling to a temperature of 0 °C, a solution of 21.93 g (65 mmol) Fmoc-OSu in 250 ml acetonitrile was added to the mixture over the course of one hour. After adjusting the reaction mixture to pH = 8 using DIEA, the mixture was warmed to room temperature and stirred overnight. The solvent mixture was removed under reduced pressure and replaced by 500 ml of concentrated, aqueous NaHCO3. This mixture was then washed three times with 250 ml ethyl acetate, acidified to a pH of 1 with concentrated muriatic acid and then extracted with again three times 250 ml ethyl acetate. The combined organic phases were dried over MgSO4 and the solvent was removed under reduced pressure to yield 17.3 g (32 mmol) of the product as a colorless oil.

Synthesis of TAMRA-NHS
5,6-TAMRA synthesis (an isomeric mixture of 5-and 6-TAMRA, from now on referred to as TAMRA) was based on Kvach et.al. 3 The procedure was performed under argon on a Schlenk line. In a typical synthesis 4.56 g 3-dimethylaminophenol (33 mmol, 1 eq.) was dissolved in 90 ml dry toluene and 7.65 g finely ground trimellitic anhydride (40 mmol, 1.2 eq.) was added under stirring. After 24 h reflux the mixture was cooled and the precipitate was washed at least 3 times with 20 ml cold toluene. The precipitate was then dissolved in 120 ml MeOH and removed by rotary evaporation to obtain a benzophenone intermediate with an average yield of 70 %. Under argon atmosphere again, 7.5 g benzophenone (22 mmol, 1 eq.) was dissolved in 170 ml dry DMF and 3.94 g 3dimethylaminophenol (28 mmol, 1.3 eq.) and 40 ml trimethylsilyl polyphosphate were added. The reaction mixture was refluxed for 3 h. After cooling, the solvent was removed under reduced pressure and the remaining residue was stirred overnight in 170 ml 5 % NaOH at room temperature. The solution was diluted with 200 ml water and neutralized with concentrated HCl to precipitate the product. The product was washed with cold water and cold ether. An isomeric mixture of TAMRA was obtained which was used without further purification. An average yield of 1.4 g (15 %) was obtained. For NHS activation, typically 50 mg TAMRA (0.11 mmol, 1 eq.) was dissolved in 12 ml dry acetonitrile and cooled to 0 °C in an ice bath. 20.2 µl dry DIEA (0.11 mmol, 1 eq.) was added and afterwards 17.9 µl DIC (0.11 mmol, 1 eq.) and 19 mg NHS (0.15 mmol, 1.3 eq.) were added. The mixture was stirred overnight in the ice bath, whereby the ice bath was allowed to come to room temperature overnight. Afterwards the acetonitrile was removed under reduced pressure. For further drying the product was freeze dried. A pink solid of the TAMRA-NHS ester was obtained and used without further purification. An average yield of 29 g (50 %) was obtained.

SPPS
All peptides were synthesized manually by Fmoc-SPPS according to Merrifield, 4 using the acid-labile 2chlorotritylchloride (2-CTC) resin. Manual solid-phase amino acid incorporation and other solid-phase manipulations were carried out in polypropylene syringes fitted with a fritted polyethylene disk CEL-053, -1016 and -2020 purchased from Roland Vetter Laborbedarf OHG. Solvents and soluble reagents were added/removed by suction/pushing of the plunger. Solutions were agitated by shaking.

Re-and Preactivation of 2-CTC Resin
2-CTC resin is used for the immobilization of the peptide during SPPS. Due to air humidity the resin can lose some of its activity during storage. Therefore, a reactivation of the resin following the procedure given by García-Martín et al. 5 can be advantageous. For the reactivation of the resin, the 2-CTC resin was lyophilized overnight to remove any moisture. Then 10 vol.-% acetyl chloride in DCM was added and shaken for one hour, washed three times with DCM and three times with DMF. Then for the preactivation of the resin 0.5 ml DIEA in 3.5 ml DMF were added and shaken for 30 minutes, leading to a change in color of the resin from yellow to deep red. After removal of the solution, the preactivated resin was washed three times with DMF.

Coupling of Fmoc-Thr(tBu)-OH to 2-CTC Resin
Fmoc-Thr(tBu)-OH (2 eq., 0.4 M) in DMF was coupled to 2-CTC resin using DIEA (4 eq.) as a base. The mixture was shaken for approx. 30 minutes. The solution was removed and the resin was washed four times with DMF. The coupling procedure was repeated a second time and the resin was washed again four times with DMF. The Fmoc group was cleaved with 20 vol.-% piperidine in DMF (1x5 min, 1×15 min) and the resin was washed 6 times with DMF. The supernatant solution of the Fmoc cleavage was collected and topped up with 20 vol.-% piperidine in DMF to 20 ml in a volumetric flask. A dilution series (10-, 100-, 1000-fold) was prepared in triplicate and the absorption at 301 nm was measured in a UV/Vis spectrometer to determine the loading of the resin. Depending on the concentration of the cleaved Fmoc, either the 100-fold or the 1000-fold diluted samples were measured. 58 mmol) using 0.57 ml DIEA (3.28 mmol, 5.6 eq.) as base. The mixture was shaken for approx. 72 hours. Thereupon, the solution was removed and the resin was washed three times with DCM. The Fmocgroup was removed as described above.

Coupling of Aminoacids (Peptide Chain Elongation)
Fmoc-Cys(Trp)-OH was attached using 2 eq. of the Fmoc protected amino acid, 2.2 eq. of DIC and 2.2 eq. of OxymaPure ® in DMF. All other amino acids were attached by employing 2 eq. of the corresponding Fmoc-protected amino acid, 2 eq. of HATU and 4 eq. of DIEA in DMF. The reaction vessel was shaken for half an hour at room temperature. All amino acids were attached by double coupling, after which the Fmoc group was removed with 20 vol.-% piperidine in DMF (1×5 min, 1×15 min) and the resin was washed 6 times with DMF.

Coupling of the Fmoc-TTDS Spacer
Attachment of the spacer was performed in the same way (2 eq. +2 eq. HATU +4 eq. DIEA) as the attachment of the amino acids.

Coupling of TAMRA
For the attachment of the dye the TAMRA-NHS ester obtained in 2.1.3 was used. The reaction was performed similarly to the attachment of the amino acids with 4 eq. of DIEA as base, but no activation agent was needed and only a single coupling step was employed. The reaction was carried out overnight. After removing the solution, the resin was washed with DMF until the supernatant showed no more signs of coloration.

Cleavage of Peptides from Resin, Deprotection and Recovery
Cleaving of peptides from the solid support and removal of side chain protecting groups was achieved via acidolysis of the dry peptide-resin using a cleavage solution consisting of TFA/TIPS/anisole/H2O (47:1:1:1, v:v:v:v). The reaction mixture was shaken for 1-2 h at room temperature before filtering and precipitation in cold MTBE and subsequent washing with MTBE and diethyl ether to yield the crude unprotected peptides. Separation of the precipitated peptides from the supernatant solutions was achieved by centrifugation.

Cyclization of Linear Peptides
Cyclization of the peptides was done by the formation of intramolecular disulfide bridges following the procedure given by Sidorova et al. 7 The linear peptides were dissolved in methanol with a peptide concentration of 0.3 mg/ml. The pH was adjusted between 6.5 and 8.0 by adding highly diluted NH3 (aq.). Then, 2.7 eq. of H2O2 were added. The reaction was left to react overnight. The reaction was stopped by addition of a few drops of acetic acid (99.5 %). The solvents were evaporated and the solid product was dissolved in 50 % ACN/H2O (v/v) and lyophilized. The cyclization was monitored by RP-HPLC and ESI mass spectrometry.

Methanol-d4 (>99.8% deuteration) was purchased from Deutero and Sigma Aldrich, the catalyst [1,4-bis-(diphenylphosphino)-butan]-(1,5-cyclooctadien)-rhodium(I)-tetrafluoroborat ([Rh(dppb)(COD)]BF4
) and D2O (99.9 %) from Sigma-Aldrich. NMR experiments were performed in a 11.7 T OXFORD 500 MHz magnet equipped with a Bruker AVANCE III HD spectrometer. The parahydrogen enrichment was performed with a parahydrogen generator from Advanced Research Systems Inc. comprising a DE204A cryostat and an ARS-4HW compressor. The cryostat is cooled to 30 K. >95 % para-enriched hydrogen was delivered into the NMR sample tube placed inside the magnet at elevated pressures and room temperature. A custom-made setup was used for bubbling directly in the tube. 8 A standard 5 mm screw-cap NMR sample tube (from Rototec Spintec, 5mm 528-TR-7) was used, closed by a cap-adapter with an in-and outlet for the gases. A thin glass capillary was attached to the inlet and immersed in the sample. The closed system was pressurized with helium, and gas flow was maintained due to a small pressure difference between the inlet and outlet at 7 bar. The gas was supplied through magnetic valves controlled by the pulse program of the NMR spectrometer, allowing not only to switch between helium and parahydrogen, but also to apply vacuum to the input/output to avoid diffusion mixing of gases in the supply tube. For maximal signal enhancement, we took care that the polarized product was formed rapidly within the time window given by T1-relaxation. The bubbling process was synchronized with the NMR pulses and was stopped 2 s before the detection of NMR spectra.

PHIP Experiments
For the PHIP experiments stem solutions of 5 mg/ml peptide (150 µl used per sample) and 3 mg/ml catalyst were prepared. They were mixed with deuterated methanol to a total volume of 690 µl to give a final 1 mM peptide concentration and three different catalyst concentrations: 0.45 mM (75 µl of stem solution), 0.9 mM (150 µl) and 1.8 mM (300 µl). The NMR tube was connected to an adapter holding a glass capillary, inserted into the magnet, filled with helium under pressure and bubbled with para-enriched hydrogen gas for 15-30 s at 7 bar. After waiting for 2 seconds a standard PHIP sequence was applied by irradiation of a 45° pulse followed by the acquisition. All PHIP-spectra were recorded as "single-shot experiments", performing one scan. Full relaxation of the polarized protons in the products was observed at least 2 minutes after the hydrogenation.
For the experiments in mixtures of methanol and water with up to 50 % of D2O, samples were prepared as before, but D2O was added instead of methanol to obtain the final sample volume and concentrations. For amounts higher than 50 % D2O, the peptide stem solution had to be prepared in D2O. This way the only methanol added originated from the catalyst stem solution. Similarly, samples were prepared in a 50 % ethanol mixture from a 2.5 mg/ml catalyst stem solution in ethanol-d6 (360 µl) and the peptide dissolved in D2O. To prevent the formation of foam, 10 µl of a 10 % TMPB solution in hexamethyldisiloxane were added as a thin layer onto the sample.

Enhancement Factors
Signal Enhancements (SE) were calculated from the absolute integrals (AInt) of the respective signal using MestreLab Research MestReNova 14.2. Since the sum of the integrals of the antiphase signals is equal to zero, AInt(PHIP) was determined as the sum of the moduli of the emission and absorption areas of the respective antiphase signal. All absolute integrals were divided by the NMR receiver gain (rg) and the number of scans (ns). In those cases where the peptide concentration deviated from 1 mM, the integral was also normalized to this value by dividing the integral by the peptide concentration (cp) in mmol/l. The signal enhancement was calculated as quotient of the normalized integral NInt(PHIP) for the polarized signal and the normalized integral NInt(therm.) for the signal in thermal equilibrium following equation: Due to the strong broadening and partial shifting of the allyl signals in relaxed spectra after the reaction ( Figure S-7), thermal signals were weak, leading to lower values for Int(therm.) and an overestimation of the signal enhancement. The further reaction of the allyl moiety to a propyl moiety also could not be ruled out completely, especially for the marker molecule A (see chapter 3.4 in SI). Therefore, the corresponding signals of the allyl variants 8 and 9, measured at the same concentration of 1 mM without undergoing the reaction, were used as external standard for Int(therm.). These integrals would correspond to the thermal integrals observed for the reaction products after hydrogenation and relaxation of the hyperpolarization, if a complete reaction of the propargyl moiety of the PHIP marker to the allyl moiety and no further reaction to the propyl moiety is assumed. The broadening and signal shift is caused by the binding of the catalyst to some of the peptide molecules either as intermediate product-catalyst complexes or by interactions with aromatic moieties of the peptide. 9,10 Table S-1 and Table S

Time Savings
In theory a SE of 1000 leads to a time saving factor of about 1 000 000. A SE of 2000 would correspond to a factor of 4 000 000. As the hydrogenation reaction before the measurement also takes time (typically 25 seconds), it needs to be considered to determine a practical time saving factor. This can be done based on the Signal to Noise Ratio (SNR

Kinetic Measurements
PHIP kinetics were measured by repeating standard PHIP measurements, described in 3.1. with 5 s bubbling time right after each other. It allows to track the maximum speed of reaction and its decline with increased total reaction time.
A second method used a single 15 second bubbling period followed by repeated 3 second delays and a short 5° pulse followed by 3 seconds of acquisition. This way we can track the polarization while hardly depolarizing the sample with the pulse (cos(5°) = 99.6 % of remaining polarization).

2D-TOCSY Experiments
For the Total Correlation Spectroscopy (TOCSY) we employed a standard dipsy2phpr experiment ( Figure S-8). The dimension Td1 was set to 1024 and 16 scans were employed for each slice. The FnMode was chosen as TPPI. The presaturation was done for d1=4 seconds at 3.9811e-5 W at the frequency of the solvent signal at 4.84 ppm. The delay d0 is incremented by 200 µs for each slice in Td1. The DIPSI-2 block was D9=80 ms long, with the delays d20=2 ms before and d21=3 ms afterwards. The concentration of the analytes was 5 mg/ml. The measurements were performed once before the reaction and two times after hydrogenation.

13 C T1-Measurements
Analogous to the T1 measurements for 1 H the T1 times of 13 C were determined by inversion recovery experiments. The measurements were done for the unprotected amino acid H-L-Tyrosine(OAllyl)-OH ( Figure S-16) in a 30 to 33 % aqueous solution of ammonia. Each spectrum was measured in 256 scans and without proton decoupling. The delay times vd were 0.2, 0.6, 1, 3, 6, 10, 30 and 60 seconds with a d1 time of 60 seconds. Measurements with delays of 120 and 300 seconds showed no further increase in the signal intensities, however, due to the noise level the integrals of those measurements were sometimes slightly smaller than those acquired with 60 seconds delay ( Figure S-17). For the linearization of the data the integral value of a simple one-pulse measurement with a d1 of 60 seconds was used for I0. The plots of the linearized datasets deviate from the linear behavior after the first six to seven data points ( Figure S-18 and Figure S-19). This could be caused by the low signal to noise ratio that leads to significant differences in the obtained integral values even after full recovery of the respective signals. Table S-4 shows the fit parameters and the resulting T1 times for the fits of the first six to seven data points for each dataset. Besides the allyl carbons A, B and C also the quaternary carbons D and E were investigated. The latter two showed the longest T1 times.

Cell Binding Assay
The cell binding assay was performed on A549 11 and HEK293 12 cells, which were tested for expression of somatostatin receptors of the type SSTR2 ( Figure S-20). The cell lines were purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ), catalogue numbers ACC 107 (A549) and ACC 308 (HEK293).

Stability of the Disulfide Bond after Hydrogenation
The stability of the disulfide bond was tested by comparing the HPLC chromatograms of the folded peptide OctF1PrgAc before and after hydrogenation with those of the folded reaction product OctF1AllAc before and after hydrogenation as well as the linear precursor of OctF1AllAc without disulfide bond. The gradient used was 20 % to 80 % of 0.1 % TFA in acetonitrile over 20 minutes at 1 ml/minute. The retention time of the linear OctF1AllAc is 11.027 minutes. The folded OctF1AllAc has a retention time of 11.560 minutes before and 11.567 minutes after hydrogenation basically remaining unchanged. This indicates no unfolding of the peptide has occurred during the reaction (Figure S-25). The retention time of OctF1PrgAc before hydrogenation is 10.991 minutes and 11.635 minutes after hydrogenation, roughly matching the retention time of the folded reaction product OctF1AllAc. This indicates that the hydrogenation reaction occurred as expected and the disulfide bond remained stable ( Figure S-26).

RP-HPLC Chromatograms
The RP-HPLC chromatograms in this section show the obtained amino alcohol and octreotide samples after the synthesis. The samples with the TAMRA fluorescence marker and spacer were purified by preparative RP-HPLC and the fractions shown here were united. Due to the two isomers of TAMRA used those samples show up to two main peaks. Only those fractions containing either one or both of the product peaks were united.